The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
Fluorescent label molecules are often used for studying of biological samples with immunochemical, cytological and histological techniques. These applications take place in aqueous solutions and one of the key requirements for the label molecule is a sufficient hydrophilicity and solubility to aqueous solutions [Hemmilä I. A., Applications of Fluorescence in Immunoassays, (ed. Winefordner J. D.) John Wiley & Sons, New York 1991]. As commonly known, the hydrophobic interactions between a fluorophore and a target molecule often leads to decrease in photophysical performance of the fluorophore. In some cases a hydrophobic label can also result in unwanted precipitation of the labelled conjugate. Hydrophilicity of the fluorescent label and also solubility of the fluorescent label in aqueous solutions can be increased by appropriate substitution of the fluorophore with a hydrophilic water-solubilizing group. These hydrophilic water-solubilizing groups include ammonium or alkali metal salts of sulfonic or carboxylic acids, quaternary ammonium salts, amino and hydroxyl groups.
For example, sulfonic acid groups have been used in Rhodamine and Cyanine dyes for improving their solubility in aqueous solutions. Derivatives of such dyes that can be used for labelling of biomolecules are also commercially available. Commercially available fluorescent labelling reagents that are soluble in aqueous solutions include Cy dyes (Amersham Pharmacia Biotech) and Alexa Dyes (Molecular Probes).
Fluorescent labelling reagents are usually attached to biomolecules via covalent linkage. For this attachment (labelling) the fluorescent labelling reagents have a functional group that can be reacted with another functional group in a biomolecule. Commonly used reactive groups include for example reactive carboxylic acid esters and anhydrides, isothiocyanates, maleimides, haloacetamides, triazines, amines, sulfonyl halides, hydrazines and alcohols. Reactive groups that are commonly used for labelling purposes can be found for example in: Haugland R. P., Handbook of Fluorescent Probes and Research Chemicals, 6th ed, Molecular Probes, Eugene, Oreg., 1996.
Dipyrrometheneboron Difluoride Dyes
A class of fluorescent dyes, dipyrrometheneboron difluoride dyes, have many desirable properties: High quantum efficiency, sharp absorption and emission bands and high absorption coefficient. These dyes have been first described by Treibs and Kreuzer, Liebigs Ann. Chem. 718 (1968) 208 and by Wories H. J. et al., Rec. Trav. Chim. Pays-Bas 104 (1985) 288. Since then dipyrrometheneboron difluoride dyes have found various applications. A wide variety of dipyrrometheneboron difluoride dyes are commercially available today (Haugland R. P., Handbook of Fluorescent Probes and Research Chemicals, 6th ed, Molecular Probes, Eugene, Oreg., 1996).
Syntheses and fluorescence properties of different derivatives of these dyes have been described in publications and patents. U.S. Pat. No. 4,774,339 describes the use of dipyrrometheneboron difluoride dyes as fluorescent labels. According to U.S. Pat. No. 4,774,339 the fluorescence properties of dipyrrometheneboron difluoride dyes are not sensitive to solvent or pH. These dyes have also narrow absorption and emission bandwidths, high quantum yield and high photostability.
The basic chromophore (I) of the dipyrrometheneboron difluoride dye has absorption and emission maxima around 500 nm.
